One-step lateral flow nonbibulous assay

ABSTRACT

A particularly efficient design for a nonbibulous lateral flow one step assay for an analyte in a biological sample is disclosed. In the improved device of the invention, three zones which are in nonbibulous lateral flow contact are employed: a sample receiving zone, a labeling zone, and a capture zone. The sample containing analyte is carried through the labeling zone and interacts with an assay label comprising visible moieties, preferably particles, which are coupled to specific binding reagent for analyte or to a competitor with analyte for a capture reagent. The flow continues into the capture zone where the visible moieties to which analyte or competitor are coupled are captured. Excess fluid is absorbed into an absorbent zone in contact with the capture zone. A positive result is obtained by visualizing the visible moieties in the capture zone. An additional improvement comprises obtaining the nonbibulous lateral flow by converting a bibulous support to a nonbibulous support through coating with a blocking agent, such a methylated BSA. Control label comprising visible moieties (preferably, visually distinguishable from those of the assay label) may also be included in the labeling zone and captured in a separate control portion of the capture zone to verify that the flow of liquid is as expected.

This application is a continuation of application Ser. No. 07/639,967,filed Jan. 11, 1991.

TECHNICAL FIELD

The invention relates to immunological and related assay methods andapparatus, especially to those for testing biological samples usingsandwich or competitive assays.

BACKGROUND ART

The literature on various forms of specific binding assays, especiallyimmunoassays, is extensive and commercial products are numerous. A largenumber of simplified and conveniently packaged assays are currentlymarketed, and patent protection has been sought for a large number ofthese.

Of particular interest in connection with the invention herein are thoseassays which are conducted using a lateral flow protocol. U.S. Pat. No.4,943,522 issued 24 Jul., 1990,incorporated herein by reference, setsforth citations which describe such devices and methods. This patentitself is directed to a lateral flow device which relies on anonbibulous support to conduct liquids from one portion of the device toanother. The present invention represents an improvement on this methodand device wherein nonbibulous lateral flow is used to conduct visiblemoieties, especially labeled particles, e.g., dyed latex, red bloodcells or liposomes capable of reacting with analyte or a competitorthereto into a capture zone for detection, using a bibulous support madenonbibulous by treatment with a blocking agent. The result is a one-stepassay which can be conducted in a very short period of time (typically,within 60 seconds), and wherein the readout is usually availableinstantaneously upon the sample contacting a capture zone.

Other disclosures of lateral flow assays have also appeared. Forexample, U.S. Pat. No. 4,861,711, issued 29 Aug., 1989 describes alateral flow assay wherein all components needed for the detection of ananalyte are embedded in a single sheet. The lateral flow is referred toas chromatographic behavior. European patent application 306,772published 15 Mar., 1989 describes a lateral flow device which comprisesa chromatographic medium wherein the zone for application of sample andthe reaction zone with an immobilized reagent capable of binding theanalyte or a label-specific binding material are separated. Britishapplication 2,204,398 published 9 Nov., 1988 and assigned to Unileverdescribes a lateral flow device wherein sample applied to the devicepicks up labeled reagent and permeates into a detection zone. Labelsinclude gold sols and colored particles. U.S. Pat. No. 4,168,146describes lateral flow through a test strip; the presence of an analyteis determined by addition of a suitable color indicator.

European application 276,152 published 27 Jul., 1988 and assigned toSymbiotics Corp. describes a bibulous matrix lateral flow device whichoperates on two separate planes. U.S. Pat. Nos. 4,094,647; 4,235,601 and4,361,537, all assigned to Thyroid Diagnostics Inc. describechromatographic strips which, when placed in developing fluid, move asample through a series of zones for binding to reagent and detection.U.S. Pat. No. 4,857,453 assigned to Syntex describes a device whereinreagents are supplied in breakable containers which are integral to thedevice.

EP 158,746 and U.S. Pat. No. 4,775,636 assigned to Janssen describe theuse of metal sols as visible detecting labels for specific bindingassays.

U.S. Pat. No. 4,703,017, assigned to Becton Dickinson, describes teststrip devices wherein binders for an analyte and/or a visible tracer areapplied to defined areas of a solid support by adsorption or covalentcoupling. After application of the binder to one or more test areas ofthe substrate, the residual binding capacity of the test substrate issaturated or blocked by treatment with one or more types of proteinswhich do not specifically bind the materials to be employed in theassay. The tracer, when bound under assay conditions to the binder or tothe analyte bound to the binder, is visible on the support withoutfurther treatment. The test strip is contacted and incubated with asample containing (or suspected of containing) analyte; a sample stripmay be provided with a plurality of test areas. U.S. Pat. No. 4,855,240,also assigned to Becton Dickinson, describes an assay wherein a sampleand a tracer as described in U.S. Pat. No. 4,703,017 are applied atdisparate locations on a flat lateral flow device.

With the exception of the first mentioned U.S. Pat. No. 4,943,522, allof the lateral flow methods heretofore discussed employ bibuloussupports. The bibulous nature of the support increases the time of theassay and permits obtaining a result only after 5 minutes or more. Theadvantages of immediate availability of results conferred by the methodsand devices of the present invention result from the conversion of thebibulous to a nonbibulous support in all regions of the matrix and fromthe design and miniaturization of the device as a whole.

DISCLOSURE OF THE INVENTION

The invention provides rapid and accurate methods for assessing thepresence or absence of analytes in biological samples and devices forthe conduct of these methods.

Thus, in one aspect, the invention is directed to an assay device fordetermination of the presence or absence of an analyte in a biologicalsample wherein the device comprises a sample receiving zone comprising asolid support capable of conducting non-bibulous lateral flow of samplein contact with a labeling zone capable of conducting nonbibulouslateral flow and containing visible moieties coupled to either aspecific binding partner reactive with the analyte or a ligandcompetitive with analyte for binding to a capture reagent, whichlabeling zone is in turn in nonbibulous lateral flow contact with acapture zone, also capable of conducting nonbibulous lateral flow andcontaining a specific binding partner for the visible moiety to whichanalyte has been attached or which is attached to competitor. The zonescan be on the same solid support or on contiguous separate supports ormatrices. The three zones are in nonbibulous lateral flow contact in thesense that they permit late al nonbibulous liquid flow between them. Thesample application, labeling and capture zones also permit liquid toflow through the zones themselves by nonbibulous lateral flow. Thecapture zone is contiguous with a means for absorbing liquid andparticles that have flowed through all three zones. The sample receivingzone may further serve to remove debris or interfering substances fromthe sample by physical entrapment without impeding the nonbibulouslateral flow.

In another aspect, the invention is directed to a method to conductone-step assays which comprises applying a sample containing orsuspected of containing an analyte to the sample receiving zone of theinvention apparatus. In still another aspect, the invention is directedto methods to prepare the apparatus of the invention, and to improvednonbibulous matrices obtained by treating bibulous matrices withblocking agents.

BRIEF DESCRIPTION OF THE DRAWINGS

(A more detailed description of the drawings follows in the Examplessection.)

FIG. 1 shows steps in the assembly of one embodiment of the lateral flowportion of the apparatus of the invention.

FIG. 2 shows the assembly of an invention device placing the lateralflow plate in a casing.

MODES OF CARRYING OUT THE INVENTION

The invention concerns improvements in assays which are conducted onsupports which conduct nonbibulous lateral flow of fluids. As defined inthe above-referenced patent U.S. Pat. No. 4,943,522, "nonbibulous"lateral flow refers to liquid flow in which all of the dissolved ordispersed components of the liquid which are not permanently entrappedor "filtered out" are carried at substantially equal rates and withrelatively unimpaired flow laterally through the membrane or support.This is distinguished from preferential retention of one or morecomponents as would occur, for example, in materials capable ofabsorbing or "imbibing" one or more components, as occurs inchromatographic separations. "Bibulous" materials include untreatedforms of paper, nitrocellulose and the like which effect chromatographicseparation of components contained in liquids which are passedtherethrough.

Bibulous materials, however, can be converted to those which exhibitnonbibulous flow characteristics by the application of blocking agents,in particular certain detergents and proteins, which obscure theinteractive forces that account for the bibulous nature of the supportsper se. Thus, nonbibulous materials include those which areintrinsically capable of conducting nonbibulous flow, such as porouspolyethylene sheets or other inert materials or can be comprised ofbibulous materials which have been blocked. Preferred blocking agentsinclude bovine serum albumin, either per se or in methylated orsuccinylated form, whole animal sera, such as horse serum or fetal calfserum, and other blood proteins. Other protein blocking agents includecasein and non-fat dry milk.

Detergent-based blocking agents can also be used. The types ofdetergents which are appropriate are selected from nonionic, cationic,anionic and amphoteric forms, and the selection is based on the natureof the membrane being blocked. Considerations which govern the selectionof the appropriate detergent blocking agent are well understood in theart. It is preferred to use detergents in combination with protein-basedblocking agents. Suitable detergents which can be used either alone orin admixture with the protein blocking agents include polyoxyethylenesorbitan alcohol detergents (i.e., the Tween series), polyoxyethylenealcohols such as Nonidet P-40 or polyoxyethylene ethers such as TritonX-100. The selection of blocking agent and formulation of the blockingcomposition is important, as the blocking must be sufficient to effectnonbibulous flow, but the modified surface must not interfere withanalyte-label-capture interaction.

One improvement which is the basis for the present invention is the useof, in the assay devices herein or in assay devices which utilizenonbibulous lateral flow in general, matrices or supports that areformed by the conversion of membranes or other supports with bibulouscharacteristics to nonbibulous membranes or supports. This is effectedthrough application of blocking solutions. While intrinsicallynonbibulous supports, such as the polyethylene sheet materialmanufactured by Porex Technologies Corporation (described in theabove-referenced U.S. Pat. No. 4,943,522), can be employed in lateralflow assays, the use of converted microporous bibulous supports ispreferred and has some advantages, such as more efficient immobilizationof a capture binding reagent and resulting increased sensitivity,improved lateral flow, and enhanced speed of detection.

To convert a bibulous support such as paper or nitrocellulose to asupport capable of effecting nonbibulous lateral flow, the originalsupport is treated with a solution of the blocking agent in an effectiveconcentration to dispose of unwanted reactivities at the surface. Ingeneral, this treatment is conducted with a blocking solution, such as aprotein solution of 1-20 mg/ml protein at approximately room temperaturefor several minutes-several hours. The resulting coated material is thenpermanently adsorbed to the surface by air-drying, lyophilization, orother drying methods.

A further improvement in the assay devices of the invention lies in thedesign of the assay itself. The invention assay devices comprise fourdistinct zones, at least three of which conduct nonbibulous lateralflow. The device is designed so that the sample containing analyte isapplied to a sample receiving zone and then flowed through a labelingzone into a capture zone. The capture zone is in turn in contact with ameans for removing excess liquid sample. In general, this will consistof an absorbent, such as filter paper or glass fiber filter, which isgenerally bibulous in nature.

Selection of the carrier porous material and its treatment is done withconsideration of the specific function each zone performs in the assaydevice.

The sample-receiving zone serves simply to begin the flow ofanalyte-containing sample and for this reason should be constructed of amaterial of low analyte retention. One means to impart this property isto impregnate the sample receiving zone with a neutral protein-blockingreagent, followed by treatment to immobilize the blocking agent (e.g.,lyophilization). An additional advantage of this treatment is increasedwetability and wicking action, speeding the transfer of the sample intothe labeling zone. The sample-receiving zone may also function as amechanical filter, entrapping any undesirable particulates present inthe sample.

The labeling zone contains visible moieties which can be detected ifaccumulated in the capture zone. The visible moieties can be simply dyesor dye polymers which are visible when present in sufficient quantity,or can be, and are preferred to be, particles such as dyed latex beads,liposomes, or metallic, organic, inorganic or dye sols, dyed or coloredcells or organisms, red blood cells and the like. Means for includingvarious dyes within liposomes are well known, and have been disclosed,for example, in U.S. Pat. No. 4,695,554 as utilized in the examplesbelow.

The visible moieties used in the assay provide the means for detectionof the nature of and quantity of result, and accordingly, theirappearance in the capture zone must be a function of the analyte in thesample. In general, this can be provided for by coupling the visiblemoieties to a ligand which binds specifically to analyte, or whichcompetes with analyte for a capture reagent in the capture zone. In thefirst approach, the visible moieties are coupled to a specific bindingpartner which binds the analyte specifically. For example, if theanalyte is an antigen, an antibody specific for this antigen may beused; immunologically reactive fragments of the antibody, such asF(ab')₂, Fab or Fab' can also be used. These visible moieties, or "test"visible moieties, then bind to analyte in the sample as the samplepasses through the labeling zone and are carried into the capture zoneby the liquid flow. When the complexes reach the capture zone, a capturereagent, which is then specific for analyte, such as an antibody orfragment thereof as set forth above, retains those coupled conjugates towhich analyte has been bound, and permits those which do not containanalyte to pass into the absorbent zone. In the second approach, thevisible moieties are coupled to a ligand which is competitive withanalyte for a capture reagent in the capture zone, most typically, othermolecules of the analyte itself. Both the analyte from the sample andthe competitor bound to the visible moieties are then carried into thecapture zone. Both analyte and its competitor then react with thecapture reagent, which in this instance is also typically specificallyreactive with analyte (and its competitor). The unlabeled analyte thusis able to reduce the quantity of competitor-conjugated visible moietieswhich are retained in the capture zone. This reduction in retention ofthe visible moieties becomes a measure of the analyte in the sample.

The labeling zone may also include "control" visible moieties which donot contain the specific binding agent or analyte competitor and whichare also carried through to a control area of the capture zone by theliquid flow. These control visible moieties are coupled to a controlreagent which binds to a capture partner specific for it and can then becaptured in a separate "control" portion of the capture zone to verifythat the flow of liquid is as expected. The visible moieties used in thecontrol may be the same or different color than those used for the testmoieties. If different colors are used, ease of reading the results isenhanced.

In all of the above, employment of the selected blocking agent togetherwith colored moieties in the labeling zone followed by theimmobilization of the blocking agent on the support (by, e.g., afreeze-drying process) is of utmost importance for improved performanceof the device. It is well known that visible moieties, especiallyparticles, aggregate upon air-drying and do not readily rehydrate incontact with a liquid sample. Therefore, absent conversion to thenonbibulous surface, instead of being transported to the capture zonewith the sample, the visible moieties will remain trapped in thelabeling zone.

As the analyte labeled with the visible moieties or the labeledcompetitor pass through the capture zone, the labeled analyte orcompetitor are bound to at least a portion of the membrane in thecapture zone (the "test" portion) through the interaction of a specificreagent applied therein which reacts directly with the analyte orcompetitor bound to the test visible moieties. If control visiblemoieties are used, provision is made for their capture in the capturezone in an area separate from that where the test visible moieties willbe bound. The control visible moieties, preferably of a different colorfrom their "test" counterparts, are provided with a control bindingagent which is specific not for analyte or the test capture reagent, butfor a material in a capture area in the capture zone which represents acontrol band. Thus they are directly detected in a separate "control"portion. For example, in the procedures exemplified below, the labelingcontrol beads include streptavidin and their control band portion in thecapture zone contains biotin, which couples to avidin specifically.Other "irrelevant" binding pairs can also be used-such asantigen/antibody reactions unrelated to analyte.

The experimental results are read in the capture zone by noting thepresence or absence of a visible signal at the location of the capturezone for the test visible moieties. The use of a control region ishelpful in that appearance of a color in the control region signals thetime at which the test result can be read, even for a negative result.Thus, when the expected color appears in the control region, thepresence or absence of a color in the test region can be noted. The useof different colors for test and control regions aids in this process.

The use of the matrix which is bibulous inherently, but convertible to anonbibulous flow characteristic is particularly useful in the creationof this capture zone. Capture reagents can be applied to the matrixbefore the application of blocking agent and immobilized in situ,obviating the need for an activation treatment as frequently requiredfor attachment of capture reagents to bibulous supports. At this stage,the bibulous nature of the matrix during the coupling of the capturereagents may be advantageous. However, the blocking/washing treatmentwhich converts the bibulous membrane to nonbibulous support providesunimpaired and speedy flow of all components of the system.

The speed of the assay, which requires less than one minute and providesin many instances essentially an instantaneous result as the sampleflows through the capture zone, is attainable because of the nonbibulousnature of the zones and of the short distance the sample must traversein each zone. Miniaturization of the diagnostic device results in theremarkable speed of the assay, well below the times disclosed in similarlateral flow assays, such as those described in U.S. Pat. No. 4,361,637;U.S. Pat. No. 4,861,711; U.S. Pat. No. 4,740,468; and European patent 0306 772 A. Miniaturization permits instantaneous results which areobservable as soon as the sample contacts the capture zone and whichoccur almost immediately or within 60 seconds of the addition of thesample to the sample receiving zone. The speed of appearance andintensity of the positive visible reaction seen depends on theconcentration of analyte in the sample. The speed of appearance of thepositive visual reaction can be adjusted to provide the optimal visualresult with concentrations of analyte of clinical importance andadjusted to suit the timing needs of the end-user.

Suitable analytes to which the method of the invention can be appliedare any for which a specific binding partner can be found. In general,most analytes of medical and biological significance can find specificbinding partners in antibodies prepared against them or fragments ofthese antibodies. Suitable analytes include soluble analytes such ashormones, enzymes, lipoproteins, bacterial or viral antigens,immunoglobulins, lymphokines, cytokines, drugs, soluble cancer antigens,and the like. These analytes include various proteins such asprotamines, histones, porphorylated proteins, nucleoproteins, and soforth such as, for example, transcortin, erythropoietin, transferrin,various globulins, thyroxin-binding globulin, the immunoglobulins ofvarious subclasses A, G, D, E, and M, various complement factors, bloodclotting factors such as fibrinogen, Factor VIII, tissue thromboplastin,and thrombin.

Also included are hormones such as insulin, glucagon, relaxin,thyrotropin, somatotropin, gonadotropin, follicle-stimulating hormone,gastrin, bradykinin, vasopressin, and various releasing factors. A widerange or antigenic polysaccharides can also be determined such as thosefrom Chlamydia, Neisseria gonorrheae, Pasteurella pestis, Shigelladysentereae, and certain fungi such as Mycosporum and Aspergillus.Another major group comprises oligonucleotide sequences which reactspecifically with other oligonucleotides or protein targets. Anextensive list of soluble analytes determinable in the method of theinvention is found in U.S. Pat. No. 3,996,345, which is incorporatedherein by reference.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of a Bead Labeled One-step Assay Device for HumanChorionic Gonadotropin (hCG )

The test device has three active components--a sample receiving zone, alabeling zone containing a particulate label and a capture zone. Thethree active portions, which communicate through nonbibulous liquidflow, are assembled so as to take advantage of a means to absorb orremove excess liquid which has been transported through these threezones, and to support the active components of the device. Theconstruction is as follows.

Preparation of the Sample Receiving Zone

The sample receiving zone is prepared from Sontara O-100 DuPont Orlonspunlace fabric. The fabric is rendered nonbibulous by saturating withmethylated bovine serum albumin (methylated BSA). The conversion tononbibulous material is achieved by treatment at 47.6 μl/cm² with a 10mg/ml solution of the methylated BSA at room temperature at fiveminutes. The pad of Sontara is then frozen at -70° C. along with alyophilization flask for at least an hour. The Sontara membrane is thenlyophilized overnight on a Virtis Freezemobile. The treated samplereceiving zone is then nonbibulous, and is cut into 10×7.5 mm rectangleswith the spunlace fibers being parallel to the longer side of the pad.

Preparation of the Labeling Zone

To prepare the labeled control beads for inclusion in the labeling zone,0.5 ml of blue latex beads initially suspended at a weight/volconcentration of 2.5% are washed twice with glycine-buffered saline(GBS) (100 mM glycine, 171 mM NaCl). The beads are sonicated for 10minutes in contact with the GBS and then microcentrifuged for threeminutes.

The pelleted beads are treated with 0.5 ml coupling solution whichconsists of 0.4 mg/ml streptavidin and 0.2 mg/ml methylated BSA in GBS.The pelleted beads are resuspended and sonicated for 10 minutes, andthen rotated overnight at room temperature.

The bead preparation is then centrifuged for three minutes and thesupernatant removed by aspiration. The bead pellet is then resuspendedin 0.5 ml of the 10 mg/ml methylated BSA. This mixture is rotated endover end for four hours at room temperature, and the bead preparationcentrifuged to recover the pellet. The supernatant is aspirated and thepellet is washed three times with bead storage solution which consistsof 1 mg/ml methylated BSA in 50 mM Tris, pH 8. The final beadpreparation is prepared in a bead storage solution at a concentration of1% solids. The resulting beads will be conducted by nonbibulous flowthrough the capture zone described below. They have been reacted so asto bind to a biotin-containing capture band.

"Test" beads for labeling analyte, containing monoclonal anti-hCG, areprepared in similar manner as follows:

One-half (0.5) ml of red latex beads (it is preferred that the controland test beads are of different colors) are washed twice with 1 ml GBSas described above by sonicating for 10 minutes and recovered bymicrocentrifugation for three minutes. To the pelleted beads is added0.5 ml of coupling solution, in this case consisting of 0.8 mg/mlmonoclonal anti-hCG and 0.2 mg/ml methylated BSA in GBS. The pellet isresuspended and sonicated for 10 minutes, and then rotated overnight atroom temperature. After centrifugation for three minutes, thesupernatant is aspirated and the bead pellet is resuspended in 0.5 ml ofthe 10 mg/ml methylated BSA and rotated end over end for four hours atroom temperature. After centrifugation and removal of the supernatant,the pellet is washed three times with bead storage solution as describedabove, and the final bead preparation is at a concentration of 1%solids.

Finally, to prepare the labeling zone containing both test and controlbeads the test beads are diluted into methylated BSA at a concentrationof 0.06% solids and the control beads into the same solution at 0.02%solids. The resultant mixture is stirred and poured onto a Sontara O-100DuPont spunlace fabric membrane at 47.6 μl/cm² The labeled pad is thenkept at room temperature for five minutes and frozen at -70° C., alongwith the lyophilizing flask for at least an hour. The resultingmembranes are lyophilized overnight on Virtis Freezemobile. Thelabel-containing pads are then cut into 10×7.5 mm rectangles with thespunlace fibers parallel to the longer side of the pad.

Preparation of Capture Zone Membrane

To prepare the capture zone membrane, nitrocellulose is renderednonbibulous as follows. Nitrocellulose obtained from Schleicher andSchuell, having a pore size of 8-12 μm, is affixed to a chart recorderand hCG capture bands are dispensed as 2-cm-spaced parallel lines usinga solution of 2 mg/ml rabbit anti-hCG (purified by protein A) in Trisbuffer using the Tri-Dac dispense system operated in the manual modewith valve pressure at 70 psi, container pressure setting at 5 psi,fluid flow at 15 sec/drop, chart speed at 500 mm/minute. These are linesreactive with the test beads. The membrane is then spotted in parallellines 0.3 cm above the previously-spotted anti-hCG capture zone with a0.6 mg/ml biotinylated rabbit gamma globulin solution dissolved in Trisbuffer. These are lines reactive with the streptavidin on the controlbeads. After air drying for 10-30 minutes, the membrane is placed onto atray containing blocking buffer (10 mg/ml methylated BSA in 50 mM Tris)for 15 minutes at room temperature. The membrane is removed and blottedand then transferred to a tray containing wash buffer (50 mM Trismaleate, pH 5.4) for 5 minutes at room temperature. The blocked andwashed membrane is then blotted, allowed to air dry, stored in adesiccator at room temperature until assembly of the device.

Assembly of the Device A 20×7.5 mm strip of the capture zone membrane,shown as component 1 in FIG. 1, is affixed centrally on an adhesivetransparency strip shown as component 5 in FIG. 1. The transparencystrip is a 700×17 mm strip of P 2200 3M transparency made adhesive withdouble-sided adhesive tape 444 (3M).

The pad containing visible label, shown as component 2 in FIG. 1, isthen affixed next to the capture zone pad with a 1 mm overlap as shownin FIG. 1. The sample receiving pad, shown as component 3, is thenplaced next to label-containing pad with 1 mm overlap, as shown in thefigure.

The device is then provided with an absorbent pad, which is a 20×7.5 mmrectangle of ED No. 939 absorbent, which is affixed to the distal end ofthe capture zone membrane with a 1 mm overlap, as shown in the figure.

The resulting test strip on the transparency backing is then coveredwith a plastic top, shown as component 6 in FIG. 2, with the length ofthe strip being centrally within the groove of the plastic top. Bothsets of capture lines are exposed in the viewing window, and the sampleapplication hole is immediately above the sample receiving pad. Finally,the bottom of the device, which is a 700×17 mm bottom strip of 1 mmthick opaque white plastic made adhesive with double-sided adhesive tape(3M) is attached to the other side of the transparency strip.

EXAMPLE 2 Preparation of a Liposome-Based Device

In a manner analogous to that set forth in Example 1, the counterpartliposome-containing device is prepared. The preparation of the samplereceiving pad, the capture zone membrane, and the assembly of thecomponents on backing is identical to that set forth in Example 1. Thelabeled liposome-containing zone pad is prepared as follows:

Liposomes are prepared according to the procedures set forth in U.S.Pat. No. 4,695,554 and contain sulforhodamine dye. Different coloreddyes are used for control and test liposomes.

For test liposomes, monoclonal anti-hCG, which has been purified fromascites, is used. Ascites is 50% SAS cut at 4° C. and desalted on a G-25column into 10 mM Tris. The antibodies are then further purified on aQ-Sepharose Fast Flow column using a salt gradient of 0-0.5 M sodiumchloride in 10 mM Tris. The fractions are monitored at 280 nm, and thepeaks collected and buffered exchanged into 1M sodium phosphate. Theantibody is diluted to 3 mg/ml and 8.5 molar equivalents of SPDP isadded. The solution is incubated for 30 minutes at 27° C. and bufferexchanged on G25 and 0.1M sodium acetate. The material is reduced with1M DTT in sodium acetate to give a final concentration of 50 mM andincubated at 27° for 25 minutes and then buffer exchanged on G25 withTris acetate buffer. The relative amounts of liposomes and antibody aredetermined assuming 8 μmol of phosphorus per mg of antibody and 1 μmolof phosphorus is supplied for 1 ml of uncoupled liposomes. The liposomesare then titrated to pH 8 with 1M Tris and coupled with antibody for twohours at 27° C.

The resulting antibody-conjugated liposomes are sized on a C6 Fast Flowcolumn diluted in liposome storage buffer to an absorbance of 0.25 unitsat 565 nm.

The liposome-containing pad is then prepared by saturating a Sontara padwith a liquid solution of anti-hCG conjugated liposomes diluted to anabsorbance at 565 nm of 0.06 at 35 μl to a 10×7.5 mm assay pad. Theliposome storage buffer for this dilution contains 10% sucrose and 10mg/ml methylated BSA. The labeling pads are kept at room temperature for5 minutes and frozen at -70° C. along with the lyophilizing flask for atleast an hour. The membranes are then lyophilized overnight in a VirtisFreezemobile.

EXAMPLE 3 Conduct of an hCG Assay

The device of Example 1 is placed flat on a benchtop and two drops ofsample at approximately 30 μl per drop are applied to the samplereceiving zone. The liquid is allowed to flow through the three zones innonbibulous lateral flow contact to the absorbent zone, with a bluecontrol band appearing in the upper portion of the viewing window inless than a minute. If hCG is present in the sample at least 50 mIU/ml,an additional red band in the hCG capture region is visible.

We claim:
 1. An assay device for detection of the presence or absence ofan analyte in a sample, said assay device providing lateral flow contactbetween three separately prepared zones, said zones comprising:(a) asample receiving zone prepared by lyophilizing a first matrix throughwhich all dissolved or dispersed components in a liquid sample flow atsubstantially equal rates, said sample receiving zone being in lateralflow contact with (b) a labeling zone prepaired by reversibly coupling alabelwhich label comprises visible moieties coupled either to a specificbinding partner reactive with the analyte or to a ligand competitivewith analyte for binding to capture reagent to a second matrix in thepresence of a methylated BSA blocking agent so that all dissolved ordispersed components in a liquid sample will flow at substantially equalrates through said second matrix, followed by lyophilizing said secondmatrix said labeling zone being in lateral flow contact with (c) acapture zone prepared by irreversibly coupling a capture reagent whichbinds to analyte to at least a portion of a third matrix followed bytreating said third matrix with a methylated BSA blocking agent so thatall dissolved or dispersed components in a liquid sample will flow insubstantially equal rates said capture zone being in fluid contact withan absorbent; wherein the presence or absence of analyte in the sampleis detectable in the capture zone within one minute of adding the sampleto the sample receiving zone.
 2. The device of claim 1 wherein saidcapture reagent is selected from the group consisting of an antibody andan immunologically reactive fragment thereof specific for analyte. 3.The device of claim 1 wherein said assay label comprises visiblemoieties coupled to a specific binding partner for said analyte so as tobind said analyte to said specific binding partner.
 4. The device ofclaim 3 wherein said specific binding partner for analyte is selectedfrom the group consisting of an antibody and an immunologically reactivefragment thereof.
 5. The device of claim 1 wherein said assay labelcomprises visible moieties coupled to a competitor of said analyte forsaid capture reagent.
 6. The device of claim 5 wherein said competitorof said analyte is additional analyte or similarly immunologicallyactive analog thereof.
 7. The device of claim 1 wherein said capturezone matrix is selected from the group consisting of nitrocellulose,nitrocellulose blends with polyester or cellulose, and nylon.
 8. Thedevice of claim 1 wherein the labeling zone further includes a controllabel comprising visible moieties coupled to a control binding reagent,and wherein said capture zone further includes a specific bindingpartner for said control binding reagent in a portion of said capturezone different from that wherein said capture reagent for said assaylabel is located.
 9. The device of claim 8 wherein said control reagentis avidin or a derivative thereof and wherein said specific bindingpartner for the control reagent is biotin.
 10. The device of claim 8wherein said assay label and control label are visually distinguishable.11. The device of claim 10 wherein said assay label and control labelare of different colors.
 12. The device of claim 1 wherein said visiblemoieties are particles.
 13. The device of claim 12 wherein saidparticles are selected from the group consisting of colored latex beads,red blood cells, metal sols and liposomes containing dye.
 14. A methodto determine the presence or absence of analyte in a sample, whichmethod comprises applying said sample to the sample receiving zone ofthe device of claim 1, so as to permit said sample to flow through thelabeling zone and the capture zone into the absorbent zone, anddetectingthe presence or absence of visible moieties in the capture zone in saidportion thereof containing the capture reagent capable of binding saidassay label.
 15. The method of claim 14 wherein said analyte is humanchorionic gonadotropin (hCG).
 16. The method of claim 15 wherein thespecific binding partner for analyte is a monoclonal antibodyimmunoreactive with hCG.
 17. The method of claim 16 wherein the capturereagent is rabbit anti-hCG.
 18. The method of claim 14 wherein thevisible moieties are particles.
 19. The method of claim 14 wherein theparticles are selected from the group consisting of colored latex beads,red blood cells, metal sols and liposomes containing dye.
 20. The deviceof claim 1, wherein said matrices of said sample receiving zone,labeling zone and capture zone have been converted from a form in whichall dissolved or dispersed components in a liquid sample do not flow atsubstantially equal rates to a form in which all dissolved or dispersedcomponents in a liquid sample flow at substantially equal rates.
 21. Themethod of claim 14, wherein said matrices of said sample receiving zone,labeling zone and capture zone have been converted from a form in whichall dissolved or dispersed components in a liquid sample do not flow atsubstantially equal rates to a form in which all dissolved or dispersedcomponents in a liquid sample flow at substantially equal rates.